EP4211229A1 - Methods for generating neural progenitor cells with a spinal cord identity - Google Patents

Methods for generating neural progenitor cells with a spinal cord identity

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Publication number
EP4211229A1
EP4211229A1 EP21865423.4A EP21865423A EP4211229A1 EP 4211229 A1 EP4211229 A1 EP 4211229A1 EP 21865423 A EP21865423 A EP 21865423A EP 4211229 A1 EP4211229 A1 EP 4211229A1
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npcs
optionally
cells
agonist
unpatterned
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German (de)
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French (fr)
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Michael G. FEHLINGS
Mohammad KHAZAEI
Christopher S. AHUJA
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University Health Network
University of Health Network
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University Health Network
University of Health Network
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Definitions

  • TITLE METHODS FOR GENERATING NEURAL PROGENITOR CELLS WITH A SPINAL CORD IDENTITY
  • the present disclosure relates to methods of generating neural progenitor cells with a spinal cord identity from starter neural progenitor cells that express Sox2, Pax6, Nestin and at least one of the brain markers (such as Otx2, Foxgl or Gbx2), or from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), as well as to compositions comprising said cells or components for making said cells and uses thereof.
  • iPSCs induced pluripotent stem cells
  • ESCs embryonic stem cells
  • NPCs neuroepithelial stem/progenitor cells
  • fbNPC forebrain forebrain NPCs
  • NPCs exposed to a temporally and spatially different gradient of morphogens gradually mature and caudalize, leading to the formation of the mid- and hind-brain.
  • spNPCs spinal cord NPCs
  • fbNPC and spNPCs are temporally and spatially located at different spectrums of this continuum (Fig. 1A), and their unique differentiation profiles likely contribute to differences in recovery observed following transplantation.
  • the fate of transplanted cells is affected by the microenvironment of the spinal cord injury site, where several cell fate determining factors, such as Shh, BMP, TGFp and Notch, are upregulated(Chamankhah et al., 2013; Chen et al., 2005; De Biase et al., 2005).
  • CNS central nervous system
  • Embryonic stem cells are PSCs capable of developing into the three primary germ cell layers in embryos.
  • ESCs formed the basis for a substantial body of knowledge around stem cell culture and cell differentiation pathways.
  • iPSCs induced pluripotent stem cells
  • iPSCs induced pluripotent stem cells
  • Their postnatal derivation eliminates ethical concerns and the potential to produce iPSCs as autologous therapies significantly reduces the risk of immune rejection.
  • NPCs neural progenitor cells
  • SCI spinal cord injury
  • PD Parkinson’s disease
  • AD Alzheimer’s disease
  • MS amyotrophic lateral sclerosis
  • stroke 2-8 multiple sclerosis
  • the regional identity of brain and spinal cord NPCs is established early in neurodevelopment (Fig. 1 ,2).
  • the neural tube is formed by infolding of the early ectodermal cells.
  • These neuroectodermal cells in neural tube expressing the neural specific markers Sox2 and Pax6 become the earliest neural stem cells. These cells, also known as neuroepithelial cells.
  • the early neural tube is a straight, elongated structure. Then the most anterior portion of the neural tube balloons into three primary regions, with the aid of transcription factors like Otx2, Lim1 , and FoxA2, that form forebrain, midbrain and hindbrain. The posterior portion of the tube matures into spinal cord 9 .
  • the most anterior/rostral fate is the first regional identity that is established 9 .
  • These cells express transcription factors such as Otx2, FoxG1 , Dlx2, Tbr1 , and Tbr2 10 ’ 11 .
  • Foxgl is expressed continuously in the postnatal and adult hippocampal dentate gyrus (DG) 12 .
  • DG dentate gyrus
  • the caudal identities will be determined by positional cues supplied by spatial gradients of patterning factors such as Wnt, FGFs, DKK1 , and FRZB.
  • These cells in the spinal cord express transcription factors, including HoxA2, HoxA3, and HoxB3 13 (Fig.2).
  • neural progenitors derived from PSCs in vitro first acquire rostral identity by default 14 , and this primitive identity can be converted to more caudal fates by various cues such as retinoic acid (RA), WNTs or FGFs , thereby mimicking the in vivo situation.
  • RA retinoic acid
  • WNTs WNTs
  • FGFs FGFs
  • Graft-host integration is enhanced by targeting the regional NPC identity
  • CST corticospinal tract
  • hiPSCs human induced pluripotent stem cells
  • spNPCs spinal cord identity neural progenitor cells
  • the inventors have for example established a culture system and use a serum-free medium and a RAR agonist such as retinoic acid (RA), to differentiate iPSCs into neural progenitor cells (NPCs) with spinal cord identity.
  • RA retinoic acid
  • spNPCs made using a method described herein can be characterized by immunocytochemistry staining and/or RT-qPCR analysis.
  • spNPCs can terminally differentiate into spinal cord specific neuronal cell types like ventral motor neurons and spinal interneurons, Renshaw cells, paragriseal, interstitial and propriospinal interneurons. These neuronal cell types cannot be generated by brain identity (e.g. unpatterned) NPCs.
  • brain NPCs can terminally differentiate to neuronal cell types of the brain like cortical, subcortical, or deep nuclear neurons, excitatory pyramidal neurons, Calbindin or CART expressing neurons, corticothalamic glutamatergic neurons and cortical cholinergic neurons that cannot be generated by spinal identity NPCs.
  • Starter neural progenitor cells that express Sox2, Pax6, Nestin and at least one of the brain markers (such as Otx2, Foxgl or Gbx2), or PSCs such as hPSCs, hiPSCs and ESCs can be used after they have been differentiated to NPCs.
  • hPSCs which were used in Examples 1 and 2, expressed pluripotency-associated genes Oct4, Sox2, and Nanog before NPC differentiation.
  • spNPCs expressed general NPC markers (Nestin, Sox2, and reduced Pax6) and also regional identity markers (e.g. Hox genes). It is demonstrated herein that spNPCs generated can be expanded in a neurosphere suspension system or monolayer cultures.
  • spNPCs were able to differentiate into cells expressing cellular markers for neurons (e.g. Fox3 and/or (3-1 ll-tubulin), astrocytes (e.g. GFAP and/or S100b), and oligodendrocytes (e.g. 01 and/or 04 and/or Olig2 and/or Olig 1 ) .
  • Starter NPCs such as unpatterned NPCs have a higher level of brain markers and lower level of spinal markers (e.g. Hox genes) than spinal identity NPCs.
  • spinal NPCs do not express detectable level of (by Immune-staining) of brain markers.
  • whole-cell patch clamp recording revealed that neurons differentiated from fbNPCs and spNPCs, exhibited electrophysiological properties of neurons, including action potentials.
  • a first aspect of the invention comprises a method of producing spinal identity neural progenitor cells (spNPCs), the method comprising: a. optionally incubating dissociated unpatterned neural progenitor cells (NPCs) in suitable culture media supplemented with, FGF2 agonist, a FGF8 agonist, optionally FGF8, to produce posteriorized NPCs expressing higher levels of at least one Hox gene, optionally HoxA4 and/or HoxA5, and lower levels of at least one of the brain markers Gbx2, Otx2 and FoxG1 compared to unpatterned NPCs; b. passaging the posteriorized NPCs and incubating the posteriorized NPCs in culture media (e.g.
  • NEM NEM supplemented with a RAR agonist, optionally retinoic acid (RA) or a RA synthetic analog, optionally EC23, and a Wnt signaling activator, optionally Wnt3a or BML- 284 hydrochloride, to produce caudalized NPCs expressing reduced levels of one or more of Gbx2, Otx2 or FoxG1 levels compared to posteriorized NPCs; c. passaging the caudalized NPCs in suitable culture media supplemented with a RAR agonist, optionally RA or a RA synthetic analog, optionally EC23; and d.
  • a ROCK inhibitor is optionally added to the culture media on day 1 after each or at least one passage.
  • the method can be initiated with unpatterned NPCs or posteriorized NPCs.
  • the unpatterned NPCs can be primed.
  • a second aspect of the invention comprises a method of priming unpatterned NPCs to stay in an ectodermal cell fate, the method comprising: a. obtaining unpatterned NPCs, the unpatterned NPCs expressing neuroectodermal markers including Pax6 and Sox1; b. priming the unpatterned NPCs of step a, the method comprising: i. adding EGF-L7 agonist, preferably EGF-L7 to culture media comprising the unpatterned NPCs of step a; and ii. optionally adding a Notch signaling activator , optionally DLL4, to the culture media, to maintain the unpatterned NPCs in the ectodermal fate.
  • Another aspect of the invention comprises a method of producing spNPCs from unpatterned NPCs, the method comprising: a. obtaining unpatterned NPCs, the unpatterned NPCs expressing neuroectodermal markers including Pax6 and Sox1; b. priming the unpatterned NPCs of step a, the method comprising: i. adding EGF-L7 agonist, preferably EGF-L7 to culture media comprising the unpatterned NPCs of step a; and ii. optionally adding a Notch signaling activator, optionally DLL4, to the culture media, to maintain the unpatterned NPCs in an ectodermal fate; and c.
  • patterning the primed unpatterned NPCs to produce spNPCS comprising: i. dissociating the primed unpatterned NPCs and incubating the primed unpatterned NPCs in suitable culture media supplemented with FGF2 agonist, a FGF8 agonist, optionally FGF8, to produce posteriorized NPCs expressing higher levels of at least one Hox gene, optionally HoxA4 and/or HoxA5, and lower levels of at least one of the brain markers including Gbx2, Otx2 and FoxG1 compared to unpatterned NPCs; ii.
  • a ROCK inhibitor is optionally added to the culture media on day 1 after each or at least one passage.
  • Another aspect of the invention comprises a method of producing spNPCs from induced pluripotent stem cells (iPSCs), the method comprising: a. producing unpatterned NPCs from the iPSCs, the method comprising: i. passaging the iPSCs and incubating said cells in iPSC culture media for about 2 days, for example 36 h to 4 days, optionally wherein the iPSC culture media comprises a TGFp inhibitor, FGF2 agonist, Wnt inhibitor, and BMP inhibitor; ii.
  • iPSCs induced pluripotent stem cells
  • a BMP inhibitor or dual SMAD inhibitors for inhibition of both TGFp and BMP pathways
  • step iii in NIM with a FGF2 agonist, optionally FGF2 or SUN 11602, for about 7 to about 11 days to produce neural rosettes, wherein the BMP inhibitor or dual SMAD inhibitors is/are removed from the media on about day 2, to produce unpatterned NPCs;
  • step a priming the unpatterned NPCs of step a, the method comprising: i. adding EGF-L7 agonist, preferably EGF-L7 to culture media comprising the unpatterned NPCs;. ii. optionally adding a Notch signaling activator , optionally DLL4, to the culture media, to maintain unpatterned NPCs in an ectodermal fate; and c.
  • patterning the primed unpatterned NPCs to produce spNPCS comprising: i. dissociating the primed unpatterned NPCs and incubating the primed unpatterned NPCs in suitable culture media supplemented with a FGF2 agonist, optionally FGF2 or SUN11602, a FGF8 agonist, optionally FGF8, to produce posteriorized NPCs expressing higher levels of at least one Hox gene, optionally HoxA4 and/or HoxA5, and lower levels of at least one of the brain markers including Gbx2, Otx2 and FoxG1 compared to unpatterned NPCs; ii.
  • a ROCK inhibitor is optionally added to the culture media on day 1 after each or at least one passage.
  • Fig. 1 depicts a schematic showing neurons that are generated from neural stem progenitor cells with different regional identity, are restricted to differentiation to specific neuronal subtypes. Forebrain derived progenitors are not able to differentiate to spinal cord specific neuronal subtypes.
  • Fig. 2 depicts a schematic showing (left) key transcription factors found in the forebrain, midbrain , hindbrain, and spinal cord regions of the CNS. (Right) Select patterning morphogens driving development of different CNS regions during embryogenesis.
  • Fig. 3 depicts a schematic showing conceptual pathway to generate spinal NPCs from human PSCs. (Bottom) Temporal exposure of key patterning morphogens in vitro modeled on developmental cues. [0028] Fig. 4 is an image depicting the morphology of neural rosettes (arrow heads) and neural tube-like structures (arrow).
  • Fig. 5A is a graph depicting gene expression profile of EGF-L7 primed NPCs (Iog2 fold change).
  • Fig. 5B is a series of two images showing the morphology of primed NPCs (bottom) are similar to un-primed/unpatterned ones (top).
  • Fig. 6A is a graph showing qPCR-based Gene expression profile of posteriorized human NPCs versus un-pattern NPCs (Iog2 fold change).
  • Fig. 6B is an image showing brightfield microscopy demonstrating the morphology of posteriorized NPCs.
  • Fig. 7A is a graph showing qPCR-based gene expression profile of caudalized human NPCs compared to posteriorized NPCs (Iog2 fold change).
  • Fig. 7B is an image showing brightfield microscopy demonstrating the morphology of caudalized NPCs.
  • Fig. 8A is a graph showing qPCR-based gene expression profile of human spinal NPCs compared to caudalized NPCs and spinal NPCs (Iog2 fold change).
  • Fig. 8B is an image showing the morphology of spinal NPCs on brightfield microscope demonstrating elongated processes and a less homogeneous appearance compared to conventional fore-brain NPCs.
  • Fig. 9A is a series of images showing a fore-brain neurosphere (left) and a spinal neurosphere (right).
  • Fig. 9B is a graph showing the results of aneurosphere assay, which demonstrate the same self-renewal potential of fb-NPCs and spNPCs after 3 passages.
  • Fig.lOA is a series of images showing the comparison of the differentiation profile between fore-brain (top panels) and spinal human PSC-derived NPCs (bottom panels) expressing neuronal marker (pill-tubulin).
  • Fig. 10B is a series of images showing the comparison of the differentiation profile between fore-brain (top panels) and spinal human PSC-derived NPCs (bottom panels) expressing oligodendrocyte marker (01).
  • Fig. 10C is a series of images showing the comparison of the differentiation profile between fore-brain (top panels) and spinal human PSC- derived NPCs (bottom panels) expressing astrocyte marker (GFAP).
  • GFAP astrocyte marker
  • 10D is a graph depicting the percent differentiation of fore-brain (top panels) and spinal human PSC-derived NPCs in cells expressing neuronal marker (pl I l-tubulin) , oligodendrocyte marker (01), or astrocyte marker (GFAP).
  • neuronal marker pl I l-tubulin
  • oligodendrocyte marker 01
  • GFAP astrocyte marker
  • Fig.11 A-C depicts voltage clamp recordings of spontaneous postsynaptic activity in fb- NPC derived neurons.
  • Fig. 11 D-F depicts voltage clamp recordings of spontaneous postsynaptic activity spNPCs derived neurons. The frequency and amplitude of postsynaptic events do not significantly differ between recordings in fb-NPCs and spNPC, indicating that much of the observed activity depends on synaptic transmission due to presynaptic action potentials.
  • Fig. 12 depicts: Generation and in vitro characterization of fbNPCs and spNPCs.
  • Fig. 12A depicts a schematic showing spatial and temporal position of fbNPCs and spNPCs along the neural tube during nervous system development.
  • Fig. 12B depicts a schematic showing fbNPCs are posteriorized, caudalized and ventralized to generate spNPCs.
  • Fig. 12C depicts images showing the morphology of fbNPCs and spNPCs in culture (GFP+).
  • Fig. 12A depicts a schematic showing spatial and temporal position of fbNPCs and spNPCs along the neural tube during nervous system development.
  • Fig. 12B depicts a schematic showing fbNPCs are posteriorized, caudalized and ventralized to generate spNPCs.
  • Fig. 12C depicts images showing the morphology of fbNPCs and s
  • Fig. 12F depicts a global view of RNA-seq analysis for differential gene expression (DEGs ) between fbNPCs and spNPCs. Heat map depicting log 10 scale of normalized TMP values (transcript pe million).
  • Fig. 12G depicts a heatmap of unsupervised hierarchical clustering of significant enriched genes involved in neural tube pattern specification.
  • Fig. 13C are images depicting the neurospheres generated from fbNPC or spNPCs, when exposed to SCI-h. Scale bar: 50pm.
  • Fig. 13C are images depicting the neurospheres generated from fbNPC or spNPCs, when exposed to SCI-h. Scale bar: 50pm. Fig.
  • Fig. 14 In vitro Differentiation Assay of NPC lines. NPCs cultured and exposed to spinal cord homogenate from uninjured (NaTve-h) or SCI-lesioned animals (SCI-h). Fig. 14Aare images depicting cells were fixed and stained for a neural progenitor cell marker (Nestin), oligodendrocyte marker (01), astrocyte marker (GFAP), or neuronal marker (pl I l-tubulin). Scale bar, 20 pm. Fig.
  • Nestin neural progenitor cell marker
  • oligodendrocyte marker 01
  • GFAP astrocyte marker
  • pl I l-tubulin neuronal marker
  • Fig. 14C depicts a graph an images showing the expression of DLL1 , the Notch activating ligand, was increased in the tissue homogenate after spinal cord injury (SCI-h) at different time points.
  • Fig. 15 Representative images characterization of the spinal cord lesion epicenter after transplantation of GFP+ fbNPCs or spNPCs.
  • Fig. 15A depicts images showing fbNPCs primarily migrated towards the lesion site to surround and partially fill the cavity, while spNPCs migrated along white matter both rostrally and caudally.
  • Fig. 15B depicts images showing higher magnification images showing the migration of fbNPC into the cavity. fbNPCs fill most of the cavity space (left). spNPCs migrate rostral and caudal to the injury epicenter along white matter tracts (right).
  • Fig. 15A depicts images showing fbNPCs primarily migrated towards the lesion site to surround and partially fill the cavity, while spNPCs migrated along white matter both rostrally and caudally.
  • Fig. 15B depicts images showing higher magnification images showing the migration of fbNPC into the cavity. f
  • Fig. 15D is a heatmap of unsupervised hierarchical clustering of significant enriched genes involved in neural tube pattern specification.
  • Fig. 16 hiPSC derived fbNPC and spNPCs demonstrate unique differentiation profiles within the chronically injured spinal cord.
  • Fig. 16A is a series of images showing transplanted cells differentiate to express markers of undifferentiated NPCs (Nestin), mature oligodendrocytes (APC), immature oligodendrocytes (Olig2), astrocytes (GFAP) and neurons (Fox3) in spNPC and fbNPC groups. Scale bars: 20 pm.
  • Fig. 17 Transplanted spNPCs contribute to remyelination following SCI.
  • Fig. 17A is a series of images showing generation of myelin is evident by colocalization of GFP+ spNPCs and MBP in close apposition to endogenous NF200 positive axons (arrowheads). Scale bar: 20 pm.
  • Fig. 17B is a series of images showing representative images of sagittal sections stained for Kv1 .2 (arrowhead) and Caspr (arrow) in spNPC. Kv1.2+ juxtaparanodal voltage-gated potassium channel and Caspr+ paranodal protein identified nodes of Ranvier.
  • Fig. 17A is a series of images showing generation of myelin is evident by colocalization of GFP+ spNPCs and MBP in close apposition to endogenous NF200 positive axons (arrowheads). Scale bar: 20 pm.
  • 17C is a series of images showing representative immunoelectron microscopic images from the GFP+ spNPCs and fbNPC. Grafted cells were detected by the black dots observed upon GFP staining (black arrows). GFP+ black dots were often observed in the outer cytoplasm of the myelin sheath in spNPCs. However, in the fbNPC group, black dots are deposited inside the axoplasm, which is ensheathed by several layers of endogenous myelin. This indicates that graft derived neurons in the fbNPC group can be myelinated. Scale bar: 200 nm.
  • Fig. 18 A-B depict the results of an antibody array showingthe different expression level of cytokines between fbNPC and spNPCs.
  • the cytokine expression profile in the conditioned media collected from cells was detected using antibody array, which allows the detection of 41 cytokines and growth factors in one experiment.
  • the fresh medium, without cell culture, was used as a background control.
  • Fig. 18C depict the results of a histomorphometric analysis using LFB and H&E staining. Representative images of the spinal cord at the lesion epicenter and 0.96 mm rostral and caudal to the area in vehicle, spNPCs and fbNPC.
  • Fig 18F is a series of representative images of very high-resolution ultrasound (VHRLIS) analyses for cavitation and Fig.
  • VHRLIS very high-resolution ultrasound
  • Fig. 18H is a series of images showing the functional vascularity was measured using power Doppler VHRLIS
  • Fig 19 fbNPCs and spNPCs can make synaptic connections endogenous cells and are invovled in electric conduction.
  • Fig. 19A is a series of images showing transmission electron micrographs of spinal cord sections at the site of transplantation demonstrating the formation of synapses between anti-GFP immunogold (black dots, white arrowheads)-labeled cells and endogenous axon terminals.
  • Fig. 19A is a series of images showing transmission electron micrographs of spinal cord sections at the site of transplantation demonstrating the formation of synapses between anti-GFP immunogold (black dots, white arrowheads)-labeled cells and endogenous axon terminals.
  • FIG. 19C is a schematic showing that to test if graft derived neurons are able to contribute to electrical transmission, we analyzed electrically evoked compound action potential (CAP) transmission across the injury site (C4 to T1).
  • Fig. 19D is a graph showing CAP over time, traces represent the average of six animals per group.
  • Fig. 19F is a graph showing CAP latency in sham, vehicle, fbNPC, and spNPC groups. Fig.
  • 19G is a graph showing CAP velocity in sham, vehicle, fbNPC, and spNPC groups.
  • Fig. 20 Functional analysis of the rats following cell transplantation.
  • Fig. 20A is a
  • Fig. 21 B is a series of images showing representative images of colocalization of GFP+ transplanted cells with Ki67. Scale bars: 20 pm. Transplanted spNPCs and fbNPCs rarely colocalize with proliferative marker Ki67.
  • Fig. 21 B is a series of images showing representative images of colocalization of GFP+ transplanted cells with Ki67. Scale bars:
  • 21 D is a series of representative images 140 days following transplantation in NOD/SCID mice. GFP positive cells of spNPC and fbNPCs dispersed in the tissue (white arrow), but there were no tumor formation in the H &.E staining.
  • Fig. 22B is a graph showing evaluation of thermal allodynia in the tail-flick test. There was no significant difference during the time course.
  • Fig. 22C is a graph showing the evaluation of mechanical allodynia in the von Frey test in the forepaw and Fig.
  • Fig. 23 is a series of images showing when the cells commit to a neuronal fate, the anterior brain and ventral spinal cord NPCs differentiate to less Ptfla expressing cells than posterior brain and dorsal spinal cord NPCs. Ptfla expressing cells will differentiate to inhibitory GABAergic interneurons.
  • Fig. 24 is a graph showing the percentage of eaten pellets over time in groups transplanted with posterior brain NPCs (posterior-NPC) , dorsal spinal cord NPCs (dorsal-NPC), anterior brain (cNPC) and ventral spinal cord NPCs (spNPC). Transplantation of posterior brain and dorsal spinal cord NPCs resulted in less improvement in functional recovery than anterior brain and ventral spinal cord NPCs.
  • posterior-NPC posterior brain NPCs
  • dorsal spinal cord NPCs dorsal spinal cord NPCs
  • cNPC anterior brain
  • spNPC ventral spinal cord NPCs
  • Fig. 25 is a series of images and a graph showing that there is no significant difference in the differentiation of cells toward oligodendrocytes (01 positive cells) in baseline differentiation medium without adding any homogenate.
  • Fig. 26A is a heatmap showing differential expression of the top 2000 genes with the highest variation between fbNPCs and spNPCs.
  • Fig. 26B depicts the results of a gene ontology enrichment of differential gene expression between fbNPCs and spNPC.
  • Fig. 26C is a graph wherein the horizontal axis shows Iog2 TPM of genes in fbNPCs.
  • Fig. 27 is a graph that shows the percentage of eaten pellets over time in fbNPC, spNPC, and vehicle groups. Transplanted cells instantly start to express and secrete trophic factors, but differentiation and integration to neural network or myelination takes time.
  • a cell includes a single cell as well as a plurality or population of cells.
  • nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art (see, e.g. Green and Sambrook, 2012).
  • the term “pharmaceutically acceptable carrier” or variations thereof is intended to include any and all solvents, media, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration and for use with cells.
  • optional examples of such carriers or diluents include, but are not limited to, buffered saline, culture media, Hanks' Balanced Salt solution, ringer’s solutions, and 5% human serum albumin and bovine serum albumin (BSA).
  • neural progenitor cell or variations thereof also referred interchangeably as neural stem cell (NSC), neural precursor cells (NPC), neural stem progenitor cells (NSPCs), neuroepithelial stem/progenitor cells (NPC) or, neuroectodermal cells (NECs), as used herein includes neural cells that express Sox2, Pax6 and Nestin and are tripotent and differentiable to neurons, astrocytes or oligodendrocytes.
  • neural progenitor cell with a spinal cord identity refers to neural progenitor cells that can terminally differentiate to spinal cord specific neuronal cell types like ventral motor neurons and spinal interneurons, Renshaw cells, paragriseal, interstitial and propriospinal interneuron cells, and which express elevated levels of spinal cord genes such Hox genes such as Hox A, B, C or D, 1-10 (e.g. A4, A5, B4, C4) in a higher amount than brain NPCs and express lower amounts of brain markers for example Gbx2, Otx2, FoxG1 , Emx2 and/or Irx2 as well as Pax6 as compared to brain NPCs.
  • Methods for producing spNPCs in vitro are provided herein.
  • Brain NPCs can differentiate to neuronal cell types of the brain like cortical, subcortical, or deep nuclear neurons, excitatory pyramidal neurons, Calbindin or CART expressing neurons, corticothalamic glutamatergic neurons and cortical cholinergic neurons that cannot be generated by spinal neurons.
  • Unpatterned NPCs refers to NPCs that have a rostral identity, and are not caudalized and/or ventralized. Unpatterned NPCs are primitive or definitive NPCs which are not yet being treated with any patterning factors like RA or Shh (and its agonists). Unpatterned NPCs express Pax6, Nestin and Sox2 and at least one of the brain markers 0TX2, F0XG1 , and GBX2.
  • the level of expression of Gbx2, Emx2 and Irx2 is lower in un-patterned NPCs as compared to mid-brain identity NPCs, and the level of expression of Hox genes (like A4, A5, B4, C4) are lower in un-patterned NPCs as compared to spinal cord identity NPCs.
  • Unpatterned NPCs can be referred to as NPCs with forebrain identity (fbNPCs), NPCs with front brain identity, or anterior brain NPCs. All of these terms refer unpatterned NPCs. They can be used interchangeable throughout this disclosure.
  • posteriorized NPCs refers to tripotent neural progenitor cells with the same differentiation profile as unpatterned NPCs.
  • the ability to form neurospheres and the proliferation rate of posteriorized NPCs are marginally higher than unpatterned NPCs.
  • Posteriorized NPCs express more Hox genes, such as HoxA4 and/or HoxA5, and have reduced expression of at least one of the brain markers such as Dlx2, Six3, LmxA1 , Gbx2, Otx2 and/or FoxG1 compared to unpatterned cells.
  • EGF-L7 agonist or variations thereof as used herein means EGF-L7, preferably human EGF-L7, (Accession number Ensembl:ENSG00000172889, MIM:608582) as well as active fragments, fusions and active splice variants thereof as well as any compound or combination of compounds, natural or synthetic, that simultaneously or in combination inhibits Notch signaling, activates EGFR (EGF receptor), inhibits ICAM-1 expression and enhances the inhibition of NF-KB activation. Combinations of molecules that activate a corresponding set of pathways can also be used instead of EGF-L7.
  • EGF-L7 means EGF-L7, preferably human EGF-L7, (Accession number Ensembl:ENSG00000172889, MIM:608582) as well as active fragments, fusions and active splice variants thereof that simultaneously inhibits Notch signaling, activates EGFR (EGF receptor), inhibits ICAM-1 expression and enhances the inhibition of NF-KB activation.
  • EGF receptor agonist means any compound or combination of compounds, natural or synthetic that binds and/or induces EGF receptor (also known as EGFR; ErbB-1 ; HER1) tyrosine kinase activity, and includes without limitation EGF, and EGF analogs as well as heparin binding EGF (HB-EGF), transforming growth factor (TGF) amphiregulin (AR) and betacellulin. Also included are EGFR activators such as NSC228155.
  • EGF epidermal growth factor
  • EGF for example human EGF
  • active fragments, fusions and splice variants e.g. fragments, fusions and splice variants that activate EGF receptor
  • RTM Cell Sciences.
  • Canton, Mass., USA Invitrogen Corporation products, Grand Island N.Y., USA, ProSpec-Tany TechnoGene Ltd. Rehovot, Israel, and Sigma, St Louis, Mo., USA.
  • FGF2 agonist or variations thereof as used herein means any compound or combination of compounds, natural or synthetic, that binds the FGF receptors that are bound by FGF2, such as FGFR1 , FGFR2, FGFR3 and FGFR4, and includes FGF2, active fragments, fusions and splice variants thereof or molecules with similar function such as SUN11602 or combinations thereof.
  • fibroblast growth factor 2 or “FGF2” or variations thereof (also known as bFGF, basicFGF, FGFb, or FGF-beta as well as heparin binding growth factor 2) as used herein refers to a member of the fibroblast growth factor family.
  • FGF2 for example human FGF2, includes active fragments, fusions and splice variants(e.g. fragments, fusions and splice variants that activate FGF receptors that are bound by FGF2) can be obtained from various commercial sources such as Cell Sciences. RTM., Canton, Mass., USA, Invitrogen Corporation products, Grand Island N.Y., USA, ProSpec-Tany TechnoGene Ltd. Rehovot, Israel, and Sigma, St Louis, Mo., USA.
  • FGF8 agonist or variations thereof as used herein means any compound or combination of compounds, natural or synthetic, that binds the FGF receptors that are bound by FGF8, such as FGFR1 , FGFR2, FGFR3 and FGFR4, and includes FGF8, active fragments, fusions and splice variants thereof or molecules with similar function such as FGF9, or FGF17 or active fragments, fusions and splice variants thereof (e.g. fragments, fusions and splice variants that activate FGF receptors that are bound by FGF8) as well as combinations thereof.
  • FGF8 agonist or variations thereof as used herein means any compound or combination of compounds, natural or synthetic, that binds the FGF receptors that are bound by FGF8, such as FGFR1 , FGFR2, FGFR3 and FGFR4, and includes FGF8, active fragments, fusions and splice variants thereof or molecules with similar function such as FGF9, or FGF17 or active fragments, fusion
  • FGF8 means FGF8 A, B or E isoform, referred to example as FGF8a, FGF8b or FGF8e and includes all naturally occurring or synthetic variants thereof, as well as mammalian FGF8 and in particular human FGF8, active fragments, fusions and splice variants thereof as well as combinations thereof.
  • FGF8 is also called androgen- induced growth factor (AIGF).
  • 740Y-P can also be replaced with high doses of FGF2 or an FGF2 agonist, for example FGF2 at a concentration of greater than 20 ng/ml up to 400 ng/ml for example at least or about 200 ng/mL.
  • FGF2 FGF2
  • 740Y-P is commercially available and can be purchased for example from Tocris Bioscience and Fisher Scientific.
  • suitable culture media means a culture media supportive of the particular cell type to be cultured.
  • a suitable culture media for neural progenitor cells or cells derived therefrom such as PSC media, NEM or NIM, for example as described herein, comprising one or more additives_for example B27 or similar additive and optionally N2 or similar additive, appropriate for the stage of cells.
  • the culture media will include non essential amino acids such as Glycine, L-Alanine, L-Asparagine, L-Aspartic acid, L-GlutamicAcid, L-Proline, L-Serine, glucose or equivalent, sodium pyruvate, Catalase , Glutathione reduced, Insulin , Superoxide Dismutase , Holo-Transferrin , Triiodothyronine (T3) , L-carnitine , Ethanolamine, D+-galactose, Putrescine, Sodium selenite, Corticosterone, Linoleic acid, Linolenic acid, Progesterone , Retinol acetate, DL-alpha tocopherol (vit E), DL-alpha tocopherol acetate , Oleic acid, Pipecolic acid, Biotin, optionally a FGF2 agonist such as FGF2 or SUN11602, EGFR agonist such as E
  • neural progenitor cells such as DMEM/F12, Neuralbasal Media etc comprising one or more of sodium pyruvate, a glutamine product such as glutamine or GlutaMAXTM, one or more antibiotics such as penicillin and/or streptomycin, a supplement such as B27 supplement without vitamin A or equivalent (e.g. without RA or RA analog) and N2.
  • Agonists can be added to the NEM depending on the stage of cell differentiation, for example one or more of an FGFR agonists such as FGF2, an EGFR agonist such as EGF and/or heparin.
  • An example of a suitable NEM is provided in Example 1.
  • Other suitable medias, supplements, antibiotics etc are known in the art and can be used.
  • Notch agonist or ““Notch signaling activator” or variations thereof as used herein includes any compound or combination of compounds, natural or synthetic including any small molecule or antibody that binds any Notch receptor and induces proteolytic cleavage and release of the Notch receptor intracellular domain. Examples include DLL1 , DLL4, Jaggedl , Jagged 2, including human and other mammalian versions thereof.
  • Wnt agonist or “Wnt signaling activator” or variations thereof as used herein includes any compound or combination of compounds, natural or synthetic including any small molecule or antibody that binds and activates a Wnt receptor. Examples include AZD2858, Wnt agonist 1 , CP21 R7 (CP21), Wnt, or BML-284 hydrochloride. Human and other mammalian versions of the biomolecule subset thereof are contemplated.
  • Wnt inhibitor or variations thereof as used herein includes any compound or combination of compounds, natural or synthetic including any small molecule or antibody that inhibits the Wnt signalling pathway. Examples include XAV939, DKK1 , DKK-2, DKK-3, Dkk-4, POCN, C59, LGK-974, SFRP-1, SFRP- 2,SFRP-5,SFRP-3,SFRP-4,WIF-1 , Soggy, IWP-2, IWR1 , ICG-001 , KY0211 , Wnt-C59, LGK974, 1WP-L6 and derivatives thereof as well as combinations thereof.
  • RAR agonist or variations thereof as used herein includes any compound or combination of compounds, natural or synthetic including any small molecule or antibody that binds and activates a RA receptor, including for example RA or an RA analog including for example EC23.
  • B27 refers to a serum free vitamin containing supplement that supports neurons and which is used with neuronal cell culture. Any such supplement that permits feeder layer independent growth can be used.
  • B27 supplement includes for example Catalase , Glutathione , Insulin , Superoxide Dismutase , Human Holo-Transferrin , T3 , L- carnitine , Ethanolamine , D+-galactose , Putrescine , Sodium selenite , Corticosterone at , Linoleic acid , Linolenic acid , Progesterone at , Retinol acetate , DL-alpha tocopherol (vit E) , DL-alpha tocopherol acetate , Oleic acid , Pipecolic acid- , and Biotin.
  • the term “rosette” or variations thereof as used herein refers to a cellular pattern of columnar cells.
  • the neural rosette is the developmental signature of neuroprogenitors in cultures of differentiating embryonic stem cells; rosettes are radial arrangements of columnar cells that express many of the proteins expressed in neuroepithelial cells in the neural tube.
  • neuroprogenitors within neural rosettes can differentiate into the main classes of progeny of neuroepithelial cells in vivo: neurons, oligodendrocytes, and astrocytes.
  • caudalized NPCs refers to NPCs having a caudal spinal cord progenitor fate and which express Sox2, Pax6 and an increased expression of Nkx6.1 relative to un-patterned NPCs and a decreased expression of Six3, Dlx2, Otx2 and FoxG1 relative to un-patterned NPCs.
  • “caudalized NPCs” express Sox2, Nestin and Pax6 with equivalent level to un-patterned NPCs, and have for example at least 75% decreased level of expression for FoxG1 , Otx2 and Gbx2, at least 25% increased expression Nkx6.1 , and have at least 25-50% increased expression of HoxA4, HoxB4 HoxC4 and HoxC5, all relative to un- patterned-NPCs.
  • the expression level of Nkx6.1 is for example at least 25% less than the expression level this gene compared to ventralized-NPCs.
  • neural induction media or “NIM” or variations thereof herein means a base media suitable for culturing neural precursor cells such as DMEM/F12 comprising one or more of sodium pyruvate, a glutamine product such as glutamine or GlutaMAXTM, one or more antibiotics such as penicillin and/or streptomycin, a supplement such as B27 supplement without vitamin A, non- essential amino acids such as Glycine, L-Alanine, L-Asparagine, L-Aspartic acid, L-Glutamic Acid, L- Proline, L-Serine, to which BMP inhibitor such as LDN193189 or Noggin, TGFp inhibitor (such as SB431542), FGFR agonist such as FGF2, optionally heparin and EGFR agonist, optionally EGF.
  • An example of a suitable NIM is provided in Example 1 in Table 1.
  • pluripotent stem cell refers to a cell with the capacity, under different conditions, to differentiate to more than one differentiated cell type, and for example the capacity to differentiate to cell types characteristic of the three germ cell layers, and includes embryonic stem cells and induced pluripotent stem cells, which are reprogrammed from somatic cells. Pluripotent cells are characterized by their ability to differentiate to more than one cell type using, for example, a nude mouse teratoma formation assay. Pluripotency is also evidenced by the expression of embryonic stem (ES) cell marker.
  • ES embryonic stem
  • iPSCs Induced pluripotent stem cells
  • hiPSCs human iPSCs
  • PSCs can for example be fetal derived, embryonic derived, or human embryonic stem cell derived.
  • stem cell refers to an undifferentiated cell which is capable of proliferation, self-renewal and giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated or differentiable daughter cells.
  • the daughter cells can for example be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential.
  • cell culture medium or variations thereof (also referred to herein as a "culture medium” or “medium”) as referred to herein is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation and optionally differentiation.
  • the cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, vitamins etc.
  • Cell culture media ordinarily used for particular cell types are known to those skilled in the art.
  • passaging refers to transferring the cultured cells from their current growth medium to a new growth medium.
  • Cells can be passaged for example according to as described in Example 1. Any suitable method of passaging however can be used. For example hiPSCs should be passaged in order to avoid overgrowth and to maintain them in an undifferentiated state. Further it may be preferable to passage iPSCs in clumps .
  • cells can be dislodged from the culture plate with the use of enzymes and enzyme cell detachment solutions such as the enzyme cell detachment solution AccutaseTM.
  • enzymes like Dispase, ReLeSR or TrypLE can also be used.
  • non-enzymatic methods like EDTA solution, can also be used..
  • N2 and non-commercial preparations thereof referred to as “hormone mix” refers to a hormone mix comprising transferrin, insulin, putrescine, selenium and prodesterone.
  • N2 can comprise 10mg/ml Transferrin, 2.5 mg/ml Insulin, 1 mg/ml Putrescine, 1 ul/ml 15 Selenium, 1 ul/ml Prodesterone.
  • N2 can be purchased commercially from Gibco (Invitrogen/Themor scientific), Sigma and others or can be prepared.
  • the method can comprise one or more features of Steps 2 and 3 in Example 1.
  • the features can be in timing, order, composition and/or selection of factors.
  • Example 1 demonstrates that suitable culture media supplemented with FGF2, EGF and 740Y-P or a synthetic agonist of 740Y-P is useful for promoting and stabilizing the identity of spinal NPCs from caudalized-NPCs.
  • Example 1 also demonstrates determining an effective range for 740Y-P. Cells prepared according to such methods, as indicated in Example 2, improve functional recovery when transplanted.
  • a first aspect of the invention comprises a method of producing spinal identity neural progenitor cells (spNPCs), the method comprising: a. optionally incubating dissociated unpatterned neural progenitor cells (NPCs), optionally primed NPCs, optionally using a cell detachment solution, in suitable culture media supplemented with FGF2 agonist and a FGF8 agonist, optionally FGF8, to produce posteriorized NPCs expressing higher levels of at least one Hox gene, preferably HoxA4 and/or HoxA5 and lower levels of at least one of the brain markers Gbx2, Otx2 and FoxG1 compared to unpatterned NPCs; b.
  • NPCs dissociated unpatterned neural progenitor cells
  • FGF8 agonist optionally FGF8 agonist
  • the caudalized NPCs of step c) in suitable culture media supplemented with a FGF2 agonist, optionally FGF2 or SLIN11602, an EGF receptor agonist, optionally EGF or NSC228155, and 740Y-P or a synthetic agonist of 740Y-P, until the identity of the NPCs are stabilized as spNPCs; wherein, a ROCK inhibitor is optionally added to the culture media on day 1 after each or at least one passage.
  • the method can be initiated using unpatterned progenitor cells or later stage cells such as posteriorized NPCs.
  • Wnt signaling activator is able to improve the activity of RA.
  • the inventors have identified that when the media in caudalizaiton step (e.g. where the posteriorized NPCs are treated with RAR agonist) is supplemented with for example Wnt3a, the increase in the level expression of Hox genes, in for example HoxA4 and/or HoxA5, is elevated in caudalized cells as compare to posteriorized cells and also the decrease in the level of expression of Gbx2, Otx2 or FoxG1 genes is boosted in caudalized cells as compared to posteriorized cells.
  • Stabilized cells have fixed identity and cannot go for example go back to other developmental identities by themselves without additional treatment. Stabilized cell will be maintained with their current identity in the proliferation or maintenance culture media. For example if they maintain their identity and gene expression profile as described herein for at least 5, at least 6 or at least 10 passages, they are considered stabilized.
  • a Rock inhibitor can be added after the first day in culture after each passage or a subset of passages, for example at least one, and optionally for one or more steps. It can be removed or left in for subsequent days if added on day 1. It can be removed for example by refreshing the media on day 2 after passage.
  • Notch inhibition is necessary for keeping cells in ectodermal fate.
  • Retinoic acid (RA) and its analogs can push cells out of an ectodermal fate.
  • Preparing spinal identity neural progenitor cells (spNPCs) requires both notch inhibition and RAR activation for example provided by RA.
  • the inventors have determined that EGFL-7 can balance these required signaling pathways. It has dual function, it inhibits Notch and at the same time does not have adverse effect on RA signaling.
  • Another aspect of the invention comprises method of priming unpatterned NPCs to stay in an ectodermal cell fate, the method comprising: a. obtaining unpatterned NPCs, the unpatterned NPCs expressing neuroectodermal markers including Pax6 and Sox1 ; b. priming the unpatterned NPCs of step a, the method comprising: i. adding EGF-L7 agonist, preferably EGF-L7 to culture media comprising the unpatterned NPCs of step a; and ii. optionally adding a Notch signaling activator, optionally DLL4, to the culture media, to maintain the unpatterned NPCs in the ectodermal fate.
  • EGF-L7 agonist preferably EGF-L7
  • DLL4 Notch signaling activator
  • another aspect of the invention comprises method of producing spNPCs from unpatterned NPCs, the method comprising: a. obtaining unpatterned NPCs, the unpatterned NPCs expressing neuroectodermal markers including Pax6 and Sox1; b. priming the unpatterned NPCs of step a, the method comprising: i. adding EGF-L7 agonist, preferably EGF-L7 to culture media comprising the unpatterned NPCs of step a; and ii. optionally adding a Notch signaling activator, optionally DLL4, to the culture media, to maintain the unpatterned NPCs in an ectodermal fate; and c.
  • patterning the primed unpatterned NPCs to produce spNPCS comprising: i. dissociating the primed unpatterned NPCs, optionally using a cell detachment solution, and incubating the primed unpatterned NPCs in suitable culture media supplemented with FGF2 agonist, a FGF8 agonist, optionally FGF8, to produce posteriorized NPCs expressing higher levels of at least one Hox gene, optionally HoxA4 and/or HoxA5, and lower levels of at least one of the brain markers including Gbx2, Otx2 and FoxG1 compared to unpatterned NPCs; ii.
  • Another aspect of the invention comprises method of producing spNPCs from induced pluripotent stem cells (iPSCs), the method comprising: a. producing unpatterned NPCs from the iPSCs, the method comprising: i.
  • the iPSC culture media comprises a TGFp inhibitor, FGF2 agonist, Wnt inhibitor, and BMP inhibitor; ii. culturing the iPSCs in iPSC culture media without a FGF2 agonist, optionally without FGF2, for about 4 days, wherein a BMP inhibitor or dual SMAD inhibitors (for inhibition of both TGFp and BMP pathways) is/are added to the culture media on about day 2, for example after about 36 h up to about 4 days; iii.
  • a Notch signaling activator optionally DLL4
  • DLL4 a Notch signaling activator
  • the caudalized NPCs of step iii) in suitable culture media supplemented with a FGF2 agonist, optionally FGF2 or SUN11602, an EGF receptor agonist, optionally EGF or NSC228155, and 740Y-P or a synthetic agonist of 740Y-P, until the identity of the NPCs are stabilized as spNPCs; wherein, a ROCK inhibitor is optionally added to the culture media on day 1 after each or at least one passage.
  • the method for producing unpatterned NPCs further comprises additional steps as described in Example 1.
  • the FGF2 agonist is FGF2, preferably human FGF2.
  • the FGF8 agonist is FGF8, preferably human FGF8.
  • the EGFR agonist is EGF.
  • the RAR agonist is RA.
  • FGF8 EGF-L7 and/or 740Y-P in the steps described, are useful for obtaining spNPCs.
  • Embryoid bodies are three-dimensional aggregates of pluripotent stem cells and express pluripotent cell markers for example Klf4 and/or Oct4.
  • Neuroectodermal cells consists of cells derived from ectoderm. One of the prominent markers of these cells is Sox1
  • the FGF8 agonist is optionally FGF8, preferably FGF8b although FGF8a, FGF8e or combinations thereof can also be used.
  • the inventors have found for example that starting from unpatterned NPCs which can be from any source, that the combination and order of factors, for example exposure to EGF-L7 followed by high concentration of FGF2 and/or FGF8, a short pulse of Wnt activation with RA, followed by RA alone and a step with 740-YP produces spNPCs.
  • Dual SMAD inhibition refers to inhibition of both BMP pathway and TGFp pathways. This can be accomplished with “dual SMAD inhibitors” which refer to an inhibitor that inhibits both pathways or a combination of inhibitors that inhibit both the BMP pathway and TGFp pathway, for example a BMP inhibitor and a TGFp inhibitor. Examples for BMP inhibitors are: Noggin, Dorsomorphin, LDN-193189, ML347, and LDN-212854 and DMH1.
  • TGFp inhibitors are: SB431532, PD169316, Galunisertib (LY2157299) or LY 3200882 as well as combinations thereof. Others are also known.
  • the iPSCs are prepared from somatic cells from a mammal, such as a human.
  • the iPSCs are human iPSCs (hiPSC).
  • the iPSCs are prepared from a subject that has sustained a spinal injury. These cells can be used to prepare autologous spNPCs which can be used for example for autologous transplantation.
  • Various molecules can be substituted, for example, for BMP inhibition: LDN-193189, ML347, LDN-212854 and/or DMH1 can be used, for TGF-p inhibition, PD 169316, Galunisertib (LY2157299) and/or LY 3200882 can be used, and for Wnt activation: KYA1797K, JW55 and/or POON can be used.
  • the unpatterned NPCs are incubated for about 3 days.
  • the posteriorized NPCs are incubated for about an additional 3 days.
  • the caudalized NPCs are incubated for about an additional 2 days.
  • the concentration of FGF2 in the culture media used for producing posteriorized NPCs is from 20ng/ml to about 150 ng/ml, for example, about 40ng/mL.
  • FGF2 agonists can be used at a concentration that provides similar effect as FGF2.
  • the concentration of FGF8 is from about 50 ng/ml to about 400 ng/ml for example about 200ng/mL.
  • FGF8 agonists can be used at a concentration that provides similar effect as FGF8.
  • the RA synthetic analog is about 0.1 pM EC23.
  • the Wnt3a concentration is about 100 pg/ml.
  • Wnt3a agonists can be used at a concentration that provides similar effect.
  • the FGF2 agonist optionally FGF2 or SUN11602
  • concentration in the culture media for passaging the NPCs until the identity of NPCs are stabilized is about 10 ng/ml.
  • the EGF concentration is about 10 ng/ml.
  • EGFR agonists can be used at a concentration that provides similar effect as EGF.
  • the 740Y-P concentration is aboutl pM.
  • the concentration of the ROCK inhibitor is about 10 pM.
  • ROCK inhibitors can be used at a concentration that provide similar effect as Y-
  • the spNPCs passaged 3 to 10 passages.
  • the concentration of EGF-L7 is about 10 ng/mL.
  • the concentration of DLL4 is about 0.5 pM
  • Notch signaling activators can be used at concentrations that provide similar effect as DLL4.
  • an agonist, activator or inhibitor is a biomolecule such as a protein, mammalian and preferably human sequences are used.
  • the method further comprises enriching and/or isolating the desired cells.
  • the spNPCs can also be further differentiated, for example, for neuronal differentiation, the spNPCs can be cultured in the absence of EGF and FGF but in the presence of BDNF, GDNF, Ascorbic acid and cAMP. As mentioned in Example 1 , differentiated cells showed neuronal morphology and expressed the neuronal marker p-lll-tubulin (Fig. 10). Astrocyte differentiation of spNPCs can be induced by exposure to BMP4 and CNTF, yielding cells with an astrocytic morphology that uniformly stained for glial fibrillary acidic protein (GFAP) (Fig.
  • GFAP glial fibrillary acidic protein
  • spNPCs can differentiated towards an oligodendroglial fate, by for example sequentially applying a Shh agonist followed by PDGF-A. After 3 weeks of differentiation, staining revealed 01-positive cells with characteristic oligodendrocyte morphology.
  • Another aspect of the invention comprises a cell population comprising spNPCs or cells differentiated therefrom produced according to methods desbribed herein.
  • the population of cells can be comprised in a composition, optionally in combination with a carrier, optionally a pharmaceutically acceptable carrier.
  • the population of cells are for use for transplantation in a recipient in need thereof.
  • the pharmaceutically acceptable carrier can be a culture media or matrix, or freezing media, optionally GMP grade or sterile.
  • Another aspect of the invention comprises an isolated cell population of primed unpatterned NPCs comprising higher expression of Nest, Pax6 and Sox2 as compared to un-primed unpatterned NPCs.
  • Another aspect of the invention comprises an isolated cell population of posteriorized NPCs comprising higher expression of HoxA4 and lower expression of Six3, Dlx2, LmxA1 , FoxG1 , Gbx2, and/or Otx2 as compared to unpatterned NPCs.
  • Another aspect of the invention comprises an isolated cell population of caudalized NPCs comprising higher expression of HoxA4 and lower expression of FoxG1 , Gbx2, and Otx2 as compared to posteriorized NPCs.
  • Another aspect of the invention comprises an isolated cell population of spinal NPCs comprising higher expression of HoxA4 and HoxA5 and less Gbx2 and Otx2 as compared to caudalized NPCs.
  • the spNPCs described herein cannot be isolated or harvested in any quantity from tissue if at all.
  • the methods described herein can be used to prepare for example autologous spNPCs, for example using fibroblasts or other somatic cells from a subject, such as a subject who has sustained a spinal injury, to prepare the isolated spNPCs.
  • the higher or increased expression is at least 1 fold change (Iog2scale) higher/increased and/or the lower or reduced expression is at least 1 fold change (Iog2scale) lower/reduced.
  • Another aspect of the disclosure comprises an isolated cell population comprising spNPCs produced according to any of the methods described herein, optionally wherein the spNPCs are derived from a patient, optionally a spinal cord injury patient, optionally wherein the isolated cell population is for transplant, optionally autologous transplant.
  • compositions comprising any isolated cell population described herien and a pharmaceutically acceptable carrier, optionally a culture media or matrix, or freezing media, optionally GMP grade, xeno free media and/or sterile.
  • any of the cell populations or compositions described herein are for use in treating a subject with a spinal cord injury or a neurodegenerative disease.
  • Another aspect of the disclosure is a method of treating a subject with a spinal cord injury or neurodegenerative disease or in the manufacture of a medicament for treating a subject with a spinal cord injury or neurodegenerative disease, the method comprising the administration of any of the isolated cell populations or the compositions described herein to treat a subject with a spinal cord injury or neurodegenerative disease or in the manufacture of a medicament for treating a subject with a spinal cord injury or neurodegenerative disease.
  • the spinal injury is a cervical, thoracic or lumbar spinal cord injury, optionally acute or chronic.
  • the neurodegenerative disease is multiple sclerosis (MS), Cerebral palsy (CP), Parkinson's disease, Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, multiple system atrophy, dementia, schizophrenia, paralysis, multiple sclerosis, spinal cord injuries, brain injuries (e.g., stroke), cranical nerve disorders, peripheral sensory neuropathies, epilepsy, prion disorders, Creutzfeldt-Jakob disease, Alper's disease, cerebellar/spinocerebellar degeneration, Batten disease, corticobasal degeneration, Bell's palsy, Guillain-Barre Syndrome, Pick's disease, and/or autism.
  • MS multiple sclerosis
  • CP Cerebral palsy
  • Parkinson's disease Alzheimer's disease
  • Huntington's disease Huntington's disease
  • Amyotrophic lateral sclerosis Friedreich's ataxia
  • Lewy body disease
  • Another aspect of the disclosure comprises a method of generating human neural stem/progenitor cells or neural precursor cell with spinal cord identity (spNPCs) comprising the following steps: a) suspending pluripotent stem cells in a culture media containing a TGFp inhibitor, FGF2 agonist, Wnt inhibitor, and BMP inhibitor; b) subjecting the cells obtained in step (a) to suspension culture in a culture media containing a Wnt inhibitor and a BMP inhibitor; c) forming an embryoid body by contacting the pluripotent human cell with an essentially serum free medium; d) culturing the embryoid body to form rosettes and neural tube-like structure and neuroectodermal cells; e) priming the neuroectodermal cells to stay in the ectodermal cell fate preferably by using EGF-L7 or its agonist(s) (for example a combination DLK1 or DAPT (to inhibit Notch), NSC228155 or betacelluin (to activate
  • Another aspect of the disclosure comprises a cell culture composition for use in a step of deriving the spinal NPCs in vitro from pluripotent stem cells, wherein the spinal NPCs express one or more detectable markers for Sox2, Pax6, Nestin or vimentin, and the spinal NPCs have the capacity to differentiate into cells of a neural lineage, comprising a base cell culture composition optionally wherein the base cell culture composition for each step is as provided in Table 1 and further comprising EGF-L7 agonist, optionally EGF-L7, for priming the neuroectodermal cells to stay in the ectodermal cell fate as in claim 27e, FGF2 agonist, optionally FGF2 and/or a FGF8 agonist, optionally FGF8, for posteriorizing the primed cells as in claim 27e), RAR agonist, optionally RA, or Wnt3a for caudaulizing the posteriorized cells as in claim 27g, and/or 740Y-P for inducing proliferation capacity as in claim 27
  • the BMP inhibitor is selected from the group consisting of Noggin, Dorsomorphin, LDN-193189, ML347, and LDN-212854, DMH1 SB431542, LDN-193189, PD169316, SB203580, LY364947, A77-01 , A-83-01 , GW788388, GW6604, SB-505124, lerdelimumab, metelimumab, GC-I008, AP-12009, AP-11014, LY550410, LY580276, LY364947, LY2109761 , SB- 505124, E-616452 (RepSox ALK inhibitor), SD-208, SMI6, NPC-30345, KI26894, SB- 203580, SD- 093, activin-M108A, P144, soluble TBR2-Fc, DMH-1 , Dorsomorphin dihydrochloride and derivatives thereof as well as combinations thereof, preferably selected from Noggin, Dorso
  • the dual SMAD inhibitors comprise a TGFp inhibitor selected from the group consisting of SB431532, PD169316, Galunisertib (LY2157299) or LY 3200882 and a BMP inhibitor selected from Noggin, Dorsomorphin, LDN-193189, ML347, and LDN-212854, DMH1 SB431542, LDN-193189, PD169316, SB203580, LY364947, A77-01 , A-83-01 , GW788388, GW6604, SB-505124, lerdelimumab, metelimumab, GC-I008, AP-12009, AP-11014, LY550410, LY580276, LY364947, LY2109761 , SB-505124, E-616452 (RepSox ALK inhibitor), SD-208, SMI6, NPC-30345, KI26894, SB- 203580, SD-093, activin-M108A,
  • the Wnt inhibitor is selected from the group consisting of XAV939, DKK1 , DKK-2, DKK-3, Dkk-4, POCN inhibitor, C59, LGK-974, SFRP-1 , SFRP- 2,SFRP- 5,SFRP-3,SFRP-4,WIF-1 , Soggy, IWP-2, IWR1 , ICG-001 , KY0211 , Wnt-C59, LGK974, 1WP-L6 and derivatives thereof as well as combinations thereof.
  • the Wnt inhibitor is POCN.
  • the Wnt inhibitor is C59 or LGK-974.
  • the pluripotent stem cell is a human pluripotent stem cell.
  • the human pluripotent stem cell is a human iPS cell or a human ES cell.
  • the culture media further contains serum or a serum substitute. In other embodiments, the culture media comprises a ROCK inhibitor.
  • Another aspect of the disclosure comprises an isolated population of human pluripotent stem cell derived spinal neural stem/progenitor cells (spNPCs) produced according to any of the methods described herein, characterized in that the spinal neural stem cells express at least one of Nestin, Sox2, Pax6, optionally wherein the spNPCs comprise a phenotype similar to a conventional neural stem cell (NSC) and at least expression of one of the Hox genes (preferably Hox A4 or Hox A5).
  • NSC conventional neural stem cell
  • At least one of the cultured cells expresses one of the detectable markers , HoxA4 or HoxA5 along with one or more detectable markers selected from Nestin, Sox2, and Pax6, wherein the amount of HoxA4 expression in the neural stem/progenitor cells is increased by at least 50% compared to the amount of HoxA4 expression in conventionally derived forebrain NPCs.
  • the cells are posteriorized cells and express more Hox genes, such as HoxA4 and/or HoxA5, and express less brain markers such as Gbx2, Otx2 and FoxG1 compared to unpatterned cells or fbNPCs.
  • the spNPCs differentiate to neurons, astrocytes or oligodendrocytes.
  • compositions comprising any of the isolated population of cells described herein and a carrier, optionally a pharmaceutically acceptable carrier, optionally a culture media or matrix, optionally GMP grade, xeno free media or sterile.
  • Another aspect of the disclosure comprises use of any of the population of spNPCs described herein or any composition described herein in the manufacture of a medicament for the treatment of a spinal cord injury of neurodegenerative disorder.
  • the neurodegenerative disorder is multiple sclerosis, cerebral palsy, Parkinson's disease, Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, multiple system atrophy, dementia, schizophrenia, paralysis, multiple sclerosis, spinal cord injuries, brain injuries (e.g., stroke), cranical nerve disorders, peripheral sensory neuropathies, epilepsy, prion disorders, Creutzfeldt-Jakob disease, Alper's disease, cerebellar/spinocerebellar degeneration, Batten disease, corticobasal degeneration, Bell's palsy, Guillain-Barre Syndrome, Pick's disease, and/or autism.
  • the spNPCs are for transplantation into a brain or spinal cord of a patient.
  • Another aspect of the disclosure is a method of treating a spinal cord injury of neurodegenerative disorder, the method comprising the administration of any population of spNPCs described herein or any composition described herein to a patient in need thereof.
  • the spNPCs are for transplantation into a brain or spinal cord of a patient.
  • the neurodegenerative disorder is multiple sclerosis, cerebral palsy, Parkinson's disease, Alzheimer's disease, Huntington's disease, Amyotrophic lateral sclerosis, Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, multiple system atrophy, dementia, schizophrenia, paralysis, multiple sclerosis, spinal cord injuries, brain injuries (e.g., stroke), cranical nerve disorders, peripheral sensory neuropathies, epilepsy, prion disorders, Creutzfeldt-Jakob disease, Alper's disease, cerebellar/spinocerebellar degeneration, Batten disease, corticobasal degeneration, Bell's palsy, Guillain-Barre Syndrome, Pick's disease, and/or autism.
  • composition comprising one or more of the cells generated using a method described herein, optionally in combination with a carrier.
  • another aspect of the invention comprises a use of any cell population described herein or composition to treat a subject in need thereof, for example a subject with a spinal cord injury or neurodegenerative disease.
  • the spinal injury may be a cervical or thoracic spinal cord injury, optionally acute or chronic.
  • the neurodegenerative disease is multiple sclerosis (MS), Cerebral palsy (CP), amyotrohic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), Friedreich's ataxia, Lewy body disease, spinal muscular atrophy, multiple system atrophy, paralysis, brain injuries (e.g., stroke), cranical nerve disorders, peripheral sensory and neuropathies.
  • a spinal cord injury or degeneration for example caused by a neurodegenerative disease, optionally MS or CP.
  • the cells or compositions may be combined with one or more neuroprotective factors, and/or the subject administered a cell population described herein may also be administered one or more neuroprotective factors, for example
  • the cells administered comprising spNPCs or cells differentiated therefrom.
  • spNPCs which as shown may express more pro-oligodendrogenic factors, which helps remyelination, are administered wtih while fbNPC or other cell types.
  • spNPCs spinal identity
  • Step 1 Generation of unpatterned NPCs from hPSCs
  • NPCs in vitro, including using “default pathway” 22 23 , or via inhibition of SMAD signaling pathway.
  • the hPSCs are cultured on a fibroblast feeder layer, they can be further expanded in feeder-free conditions for 3-4 passages prior to induction of neural progenitors. This action acclimates the cells, improving culture quality and yield.
  • hPSC small clumps of hPSC will be cultured on ultra-low adherent dishes in hPSC culture media (without FGF2) and neural induction media for 7 days. During this period, hPSCs grow to cell aggregates which are called EBs.
  • NIM Neuroectodermal Medium
  • FGF2 signaling is necessary for the polarization of rosettes. Fibroblast growth factor 2 (FGF2) is then added to guide the transition of the neuroectodermal cells into rosette structures.
  • FGF2 Fibroblast growth factor 2
  • NEM is for transitioning NPCs to produce NPC that express Nestin, Sox2, and Pax6
  • Alternate methods of passaging to Accutase dissociation include using 0.5 mM EDTA in Dulbecco’s PBS without MgCh, CaCh, or ReLeSR.
  • ReLeSR selectively lifts only iPSC cells, leaving differentiated cells on the plate. This allows for quick and easy selection for regular iPSC culture as well.
  • Dorsomorphin and SB431532 block the BMP and TGF-p signaling pathways, which has been shown to improve the efficiency of neural induction to greater than 80% of total cells 14 .
  • EBs Cell aggregates in the form of EBs should be observed by day 5. EBs simulate the endogenous conditions under which pluripotent hPSCs transition into neuroectodermal cells.
  • Neural Rosette Selection Reagent Stem Cell Technologies
  • a brief incubation 3-5 min
  • Dispase, tapping and a PBS wash to lift neural rosettes.
  • Neural Rosette Selection Reagent had been found to be sub-optimal for selectively lifting neural rosettes of monolayer differentiation cultures, so use in only EB cultures is recommended.
  • PLL 0.1 mg/ml solution
  • laminin pre-coated plates at 1 x10 5 cells/cm 2 . Avoid re-plating at lower densities, as this promote undesired differentiation and loss of secondary rosette formation.
  • Laminin-511 (but not -332,-111 , or -411) is preferred over other ECM replacements, such as Matrigel or Geltrex due to it being growth-factor free, which may interfere with the differentiation process.
  • the culture should contain isolated NPCs that express Nestin, Pax6, and Sox2, but not Oct4.
  • NEM Use a flame-polished Pasteur pipette to pipette the media up and down 10-20 times, or until separation into single cells is observed. Plate the suspension at 10 cells/pL on ultra-low adherent plates in NEM.
  • hPSC-NPCs generated using this method will, by default, express FoxG1 , Gbx2 and Otx2, markers of forebrain to midbrain identity. Cells will not express HoxC4, a marker of spinal identity in NPCs.
  • Step 2 Keeping the NPCs in the ectodermal cell fate
  • Step 1 Bone Morphogenetic Protein 4 (BMP4) signaling was inhibited by BMP inhibitor Dorsomorphin., LDN193189 (LDN) or Noggin can also be used, and TGFp was inhibited by SB431542 (SB) to prevent mesodermal and endodermal differentiation.
  • BMP4 Bone Morphogenetic Protein 4
  • DMP inhibitor Dorsomorphin.
  • LDN193189 LDN193189
  • Noggin TGFp was inhibited by SB431542 (SB) to prevent mesodermal and endodermal differentiation.
  • SB SB431542
  • RA Retinoic Acid
  • EGF-L7 10 ng/mL
  • EGF-L7 interacts with all the four Notch receptors (Notch1-4) and inhibits/competes with Jagged 1 and Jagged2 proteins (not DLL4) for their interaction with Notch receptors 29 .
  • EGF-L7 knockdown stimulates the Notch pathway and EGF-L7 over-expression inhibits the Notch pathway. While NPCs are actively proliferating, Notch signaling contributes to the maintenance of the undifferentiated state.
  • EGF-L7 activates EGF-receptor, but it is less potent than EGF and modulates Notch signaling which reduce the hyper-proliferation of NPCs.
  • DLL4 Delta-Like 4; a Notch signaling activator
  • Fig 5 the level of expression of neural progenitor genes like Nestin and Pax6
  • NMPs neuromesodermal progenitors
  • Step 3 Patterning NPCs towards a spinal cord-specific identity: [00198] To generate spNPCs, we patterned cells using a stepwise treatment of morphogens 33 . Patterning of primed NPCs towards a spinal cord identity is modelled on the developmental cues that are involved in the formation of the spinal cord during embryogenesis.
  • the region of the neural plate giving rise to the spinal cord is specified in an FGF-dependent manner.
  • FGFs including FGF3, FGF4, FGF8, FGF13, FGF18, are involved in spinal cord specification.
  • FGF3 FGF4, FGF8, FGF13, FGF18.
  • In vitro experiments have shown that exposure of neural tissue to increasing FGF levels results in progressively elevated levels of HOXC6, HOXC8, HOXC9, or HOXC10 34 ’ 35 .
  • several signaling pathways influence FGF8 expression.
  • the Wnt and Shh pathways which are active in the caudal region of the neural tube, can themselves increase FGF8 levels 3637 .
  • Table 2 contains a list of material that can be used for this protocol.
  • FGF2 from 20ng/ml to 150 ng/ml
  • FGF8 from 50 ng/ml to 400 ng/ml
  • caudal cells are exposed to select FGFs for longer periods of time than rostral cells they are involved in regionalization of the spinal cord along the rostral-caudal axis.
  • FGF8 is more broadly expressed. Expression of FGF8 continues for several days but declines toward the final stages of somitogenesis and the cessation of axis elongation 3839 . Treatment with FGF8 at this concentration and time period results in posteriorization of the cells.
  • the posteriorized NPCs produced at the end of this stage express more Hox genes, such as HoxA4, and have reduced expression of at least one of the brain markers such as Gbx2, Otx2 and FoxG1 compared to unpatterned cells (Fig. 6).
  • Posteriorized NPCs are equally tripotent with the same differentiation profile as un-patterned NPCs. The ability to form neurospheres and the proliferation rate of posteriorized NPCs are marginally higher than un-patterned NPCs.
  • RA retinoic acid
  • EC23 the synthetic retinoid analog
  • FGF and RA signaling are not sufficient (alone or together) to induce caudal characteristics in neural cells grown in vitro and Wnt signaling (Wnt3a) is further required to specify neural cells to a caudal identity 42 .
  • the self-renewal capability spNPCs is comparable to that of fbNPCs, as it was determined using a clonal analysis (Fig.9). Neurospheres were mechanically dissociated to a single cell suspension and plated at clonal density in the neurosphere assay (Fig. 9). No significant difference was found in the self-renewal ability of fbNPCs or spNPCs during 3 passages.
  • spNPCs The developmental potential of spNPCs are comparablee to that of fbNPCs as assesses by analysing their capacity for differentiation into the three main neural lineages.
  • NPCs were cultured in the absence of EGF and FGF but in the presence of BDNF, GDNF, Ascorbic acid and cAMP. Differentiated cells showed neuronal morphology and expressed the neuronal marker
  • Astrocyte differentiation of fbNPCs and spNPCs was induced by exposure to BMP4 and CNTF, yielding cells with an astrocytic morphology that uniformly stained for glial fibrillary acidic protein (GFAP) (Fig. 10).
  • GFAP glial fibrillary acidic protein
  • fbNPCs and spNPCs To compare the ability of fbNPCs and spNPCs to differentiate towards an oligodendroglial fate, we employed a protocol that sequentially applies a Shh agonist followed by PDGF-A. After 3 weeks of differentiation, staining revealed 01-positive cells with characteristic oligodendrocyte morphology.
  • the spontaneous synaptic activity of neurons derived from spNPCs is comparable to that of fbNPCs as determined using whole-cell patch-clamp recordings that demonstrated both lines had similar inward sodium currents and were able to generate action potentials. Furthermore, the amplitude of sodium currents and the firing properties of neurons were not significantly different between the two groups (Fig. 11).
  • This study seeks to determine how the differentiation state of transplanted neural stem/progenitor cells is modulated by the spinal microenvironment to promote recovery.
  • NPCs at different stages of development fbNPC vs spNPCs
  • fbNPC mainly differentiated into neurons
  • spNPCs mainly differentiated to myelinating oligodendrocytes.
  • the unique differentiation profiles were mainly due to differential Pax6 expression between the two lines, and were affected by activation of Notch signaling in the injured spinal cord microenvironment.
  • Transplantation of both NPCNPCS lines resulted in neurobehavioral recovery, including improvements in forelimb grip strength and measures of forelimb/hindlimb locomotion, as assessed by Catwalk.
  • fbNPC produced their effects in functional recovery through differentiation to neurons, which migrated towards the cavity and formed a cellular bridge.
  • spNPCs produced their effects through remyelination. Both lines provided trophic support for tissue preservation and regeneration.
  • GFP-positive-hiPSCs generated by non-viral, piggyBac transposon-mediated reprogramming (Hussein et al., 2011), were used to establish fbNPC and spNPCs by mimicking key morphogenic cues and replicating developmental neural tube patterning in vitro (Fig.12A-C). A combination of different growth and patterning factors were applied to induce the generation of fbNPC and spNPCs. For the establishment of fbNPC, a dual SMAD inhibition method was used(Varga et al.).
  • fbNPCs were generated using dual SMAD inhibition, caudalized and ventralized using agonists for retinoic acid (RA) and Sonic hedgehog (Shh), and maintained in a culture media to preserve their ventral spinal cord identity using the method described in Example 1.
  • a comparative gene expression analysis revealed that the expression of pluripotent cell markers (Oct4, Nanog) was decreased, whereas expression of neural cell markers (Sox2, Pax6 and Nestin) was increased in both lines compared to the original hiPSCs.
  • the expression of Nestin and Sox2 was comparable between fbNPC and spNPCs (Fig.12D), but fbNPC were observed to express 2.2 fold more Pax6, than spNPCs (Fig.12D).
  • Gene expression profiling of fbNPC showed higher expression levels of transcription factors Otx2 and FoxG1 , which are markers of anterior identity cells, as compared to spNPCs.
  • spNPCs expressed increased levels of Nkx2.2, Nkx6.1 , HoxA4 and HoxA5 transcription factors, which represent spinal cord identity.
  • Fig.12 E To compare the global transcriptome of fbNPCs and spNPCs, we performed RNA-seq analysis (Fig. 12F). Despite the considerable similarity of gene expression patterns in fbNPCs and spNPCs, we identified some important differences. There was an increased expression of spinal cord specific Hox genes and decreased expression of brain related patterning transcription factor in spNPCs as compared to fbNPCs (Fig. 12G).
  • neurons, astrocytes and oligodendrocytes arise from common neuro epithelial progenitor cells in a process guided by the dynamic interplay of environmental signals(Silbereis et al., 2016). However, after SCI, these developmental cues are not present and different environmental factors are expressed.
  • Fig. 13A-C Neurospheres were mechanically dissociated to a single cell suspension and plated at clonal density in the neurosphere assay.
  • Fig. 13A-C There was no significant difference in the self-renewal ability of fbNPC and spNPCs when treated with NaTve-h, as the number of neurospheres generated from both lines was not significantly different (Fig. 13A-C).
  • fbNPC when treated with SCI-h, fbNPC generated significantly more neurospheres and demonstrated greater average neurosphere size as compared to spNPCs, suggesting that the proliferation rate of the fbNPC forming neurospheres was higher than spNPCs after exposure to SCI-h (Fig. 13A-C). This was further confirmed using a Brdll assay (Fig. 13D).
  • fbNPC differentiated to more neurons (31.7 ⁇ 2.0 %,) compared to spNPCs (20.5 ⁇ 1.9 %, p ⁇ 0.5), while spNPCs differentiated to more 01 expressing oligodendrocyte (fbNPC; 31.7 ⁇ 2.0 % v.s. spNPCs; 20.5 ⁇ 1.9 %, p ⁇ 0.5).
  • fbNPC differentiated to more neurons (31.7 ⁇ 2.0 %,) compared to spNPCs (20.5 ⁇ 1.9 %, p ⁇ 0.5)
  • spNPCs differentiated to more 01 expressing oligodendrocyte fbNPC; 31.7 ⁇ 2.0 % v.s. spNPCs; 20.5 ⁇ 1.9 %, p ⁇ 0.5
  • culturing in the presence of SCI-h had a distinct effect on fbNPC compared to spNPCs.
  • Notch signaling is a pathway involved in binary cell fate decisions as well as induction or enhancement of terminal differentiation.
  • Notch signaling is first used to induce the self-renewal and proliferation of cells.
  • Notch induces the differentiation of cells to a glial fate(Grandbarbe et al., 2003; Namihira et al., 2009; Tanigaki et al., 2001). Therefore, Notch signaling is a potential candidate responsible for the observed developmental stage differences in differentiation after exposure to SCI-h.
  • DLL1 expression reaches its maximum 2 weeks after injury and begins to decrease around 2 months post injury.
  • the distinct effect of Notch on developmentally different neural progenitors is correlated with their level of Pax6 expression(Sansom et al., 2009).
  • Pax6 expression levels are higher in fbNPC compared to spNPCs.
  • the Pax6-regulated activity in neural stem cells is highly dose sensitive, with increasing Pax6 levels driving the system towards neurogenesis.
  • Relative levels of Pax6 and Notch signaling are factors in a dynamic balance that controls whether neural stem cells self-renew, differentiate to neurons, or generate glial progenitor cells.
  • Notch signaling increased Pax6 activity suppresses neurogenesis and promotes self-renewal, in part by repression of Neurog2.
  • decreased levels of Pax6 in the presence of Notch signaling result in gliogenesis.
  • fbNPC and spNPCs survived, migrated and differentiated in the injured spinal cord.
  • T-cell deficient RNll rats received a C6/7 cervical SCI followed by cell transplantation at 2 weeks postinjury.
  • Transplanted cells (GFP + ) were found in both the white and gray matter.
  • Grafted fbNPC were located mainly around the lesion epicenter (Fig. 15A,B) and showed a tendency to migrate towards the injury epicenter and to fill the cavity (Fig.15A, B).
  • spNPCs migrated as far as 7 mm rostral and caudal from the epicenter, and predominantly migrated along white matter tracts (Fig 15 A, C).
  • the chemotactic response of neural stem cells to the injury site is highly related to their differentiation status and the type of lesion(Filippo et al., 2013; Imitola et al., 2004).
  • Chemokines such as CXCL12
  • CXCL12 receptors CXCR4 or CXCR7(Chen et al., 2015; Imitola et al., 2004).
  • fbNPC express higher levels of chemoattractant receptors CXCR4 and CXCR7, cell adhesion molecules (e.g. CD44) and integrins (e.g. ITGA4), as compared to spNPCs, which may explain this chemotactic response (Fig.15D)(Filippo et al., 2013; Imitola et al., 2004).
  • both fbNPC and spNPCs differentiated into neurons, astrocytes, and oligodendrocytes in vivo, however, a proportion of cells in both lines remained in an undifferentiated Nestin positive state.
  • the number of nestin positive cells for the fbNPC line was more than two times greater than spNPCs (fbNPC : 31.2 ⁇ 7.1 and spNPCs : 13.21 ⁇ 4.5 %) (Fig. 16A,B) in keeping with observed in vitro results after SCI-h exposure.
  • the percentage of APC positive mature oligodendrocytes was significantly higher in transplanted spNPCs (54.23 ⁇ 5.24%) as compared with fbNPC (30.4 ⁇ 2.14%; p ⁇ 0.05). Similarly, more immature Olig2 positive oligodendrocytes were observed in the spNPC group (spNPCs: 39.9 ⁇ 7.9 and fbNPC: 18.3 ⁇ 3.7 %).
  • the presence of GFAP positive astrocytes was not significantly different among transplanted spNPCs (25.4 ⁇ 5.1 %) as compared to fbNPC (16.2 ⁇ 4.2%).
  • Fox3 positive neurons were significantly more abundant in transplanted fbNPC (22.6 ⁇ 1.9%) than spNPCs (6.5 ⁇ 0.8%) (Fig. 16A,B).
  • the neurons derived from transplanted cells need to be myelinated.
  • graft derived oligodendrocytes can contribute to the remyelination of denuded axons.
  • MBP myelin basic protein
  • NF200 myelin basic protein
  • trophic factors have pro-neurogenic, pro-axonogenic, as well as various anti- apoptotic and pro-angiogenic effects, which preserve endogenous tissue(Ruff et al., 2012).
  • fbNPC and spNPCs a human growth factor antibody array to analyze the secretion of different growth factors between fbNPC and spNPCs. Both lines showed expression of different fibroblast growth factor (FGF) isoforms at an increased level. FGF isoforms have been linked to survival and neurite outgrowth in certain neuronal subtypes(Pataky et al., 2000).
  • FGF fibroblast growth factor
  • pro-neurogenic trophic factors such as NT3, NGF, and GDNF
  • NT3, NGF, and GDNF pro-neurogenic trophic factors
  • GDNF pro-neurogenic trophic factors
  • trophic factors that induce differentiation and proliferation of glial lineages like PDGF and TGF isoforms, were increasingly expressed by spNPCs, while pro-angiogenic factors like VEGF were greatly expressed by fbNPC (Fig. 18A,B).
  • spNPCs and fbNPC groups were significantly smaller compared with vehicle (vehicle: 3.82 ⁇ 0.45 mm3, spNPCs : 1.74 ⁇ 0.20 mm3, fbNPC : 1.89 ⁇ 0.26 mm3, p ⁇ 0.01) (Fig. 18E).
  • VHRLIS imaging is capable of generating planar full-depth images and 3D reconstruction volumes of cavitation in situ. This helps to overcome difficulties with tissue shrinkage that occur during routine histological processing(Soubeyrand et al., 2014).
  • vehicle treated animals vehicle treated animals
  • spNPC-derived neurons make synaptic connections with endogenous cells and enhance electric conduction [00240]
  • the neurons that are differentiated from transplanted cells must form synaptic connections with endogenous cells and integrate into local networks to promote functional recovery.
  • SYN1 Synapsin I
  • Fig. 19 we assessed whether gold-labeled GFP+ cells formed synaptic connections with labelnegative endogenous cells (Fig. 19). Both fbNPCs and spNPC were able to form synaptic connections.
  • fbNPCs express higher levels of chemoattractant receptors CXCR4 and CXCR7, cell adhesion molecules (e.g. CD44) and integrins (e.g. ITGA4), as compared to spNPCs, which may explain this chemotactic response. (Fig 26).
  • NPC derived neurons could potentially form synapses with endogenous neurons and improve neural circuit conduction(Lu et al., 2012, 2014) by bridging and relaying supraspinal neurons from the cortex to their spinal targets(Bonner and Steward, 2015).
  • the NPC derived oligodendrocytes could potentially contribute to remyelination.
  • spNPCs mainly differentiated to Olig2 and/or APC positive oligodendrocytes. These cells could differentiate to MBP positive myelinating oligodendrocytes (Fig.
  • cystic cavities form as a result of cell death and the clearance of damaged tissue by phagocytotic cells(Dusart and Schwab, 1994; Liu et al., 1997). This damage to the spinal cord significantly affects functional recovery.
  • transplanted NPCs mainly fbNPC
  • This cellular bridge provides a structural substratum that allows injured axons to grow into a permissive environment. Migration towards the cavity (pathotropism) is very important for forming this cellular bridge.
  • chemoattractants like CXCL12 (SDF1), are expressed by the injured site post-SCI, and attract cells that express CXCL12 receptors, CXCR4 or CXCR7(Chen et al., 2015; Imitola et al., 2004). It has been shown that neural stem/progenitor cells at their early stages of development show higher expression of these receptors and a higher degree of pathotropism (Chang et al., 2013; Ferrari et al., 2012). This is in concordance with our results showing that early stage NPCs (fbNPC) express higher levels of CXCR4 and CXCR7 than spNCEs, and demonstrate a higher tendency to migrate towards the cavity and form a cellular bridge.
  • fbNPC early stage NPCs
  • transplanted NPCs provide trophic support to the injured tissue. Secretion of trophic factors promotes tissue repair by preventing tissue damage and enhancing the survival of endogenous neural cells. It also re-establishes the functional interactions between neural and glial cells(Ruff et al., 2012). Both NPC lines secreted elevated levels of trophic factors like FGF, NT3, NGF, and GDNF, which are involved in the survival and regeneration of injured tissue.
  • composition of secreted growth factors was slightly different between the cell types, with spNPCs expressing more pro-oligodendrogenic factors, which helps remyelination, while fbNPC expressed more pro-angiogenic factors that help restore vascularity in the injured tissue.
  • spNPCs expressing more pro-oligodendrogenic factors, which helps remyelination
  • fbNPC expressed more pro-angiogenic factors that help restore vascularity in the injured tissue.
  • a proportion of the functional recovery observed in fbNPC transplanted animals could be attributable to providing a permissive cellular bridge in the cavity as well as integration and connection of fbNPC derived neurons into the host neural network. Conversely, a large proportion of spNPCs’ effects appear to be the result of enhanced remyelination. Both mechanisms are important for functional recovery. Further experiments are required to determine the synergistic effect of transplanting a combination of both fbNPC and spNPCs.
  • hiPSCs line PB1-53 were differentiated to NPCs using dual SMAD inhibition in monolayer culture with some modifications(Varga et al.).
  • fbNPC were maintained in DMEM:F12 supplemented with N2, B27-VA media with FGF2 (10ng/ml) and a combination of inhibitors targeting TGFp (SB431542) and WNT signaling (CHIR99021)(Payne et al., 2018; Varga et al.).
  • spNPCs we patterned cells with stepwise treatment with patterning factors (Lippmann et al., 2015).
  • cells were cultured in DMEM:F12 media supplemented with B27, N2, FGF2 (20ng/ml), FGF8 (200 ng/ml) for three days(Lippmann et al., 2015). Cells were then caudalized by supplementing the culture with 0.1 pM retinoic acid agonist EC23 for an additional 5 days. Cells underwent ventralization by treatment with 1 pM sonic hedgehog (Shh) agonist Purmorphamine for 3 days. At this stage cells showed a ventral spinal cord identity.
  • spNPCs were maintained in media consisting of B27, N2, FGF2 (10ng/ml), EGF (10ng/ml) and 740Y-P (1 pM) for three passages prior to transplantation. During passaging, 10 pM Rock inhibitor (Y-27632) was added on day 1. Further details are provided in Example 1. mRNA expression profiling
  • RT-PCR Quantitative RT-PCR was used to examine the expression profile of fbNPC and spNPCs.
  • mRNA was isolated using the RNeasy Mini Kit (Qiagen74104).
  • a Nanodrop spectraphotometer was used to evaluate the concentration and purity of the mRNA.
  • cDNA was synthesized using a SensiFASTTM cDNA Synthesis Kit (Bioline 65053) with random hexamere primers.
  • RT-PCR was performed using TaqMan design primers with SensiFAST Probe Hi-ROX master mix (Bioline 82020) under recommended thermocycling parameters on a 7900HT Real time PCR system. Samples were run in triplicate. Values were normalized to the GAPDH housekeeping gene. For the examination of the gene expression levels, results were normalized to GAPDH and to the reference cells. The gene expression level was calculated using the 2 -AACT method.
  • NPCs were cultured on their maintenance media without growth factors (no FGF2 for fbNPC and no FGF2/EGF for spNPC) and treated with 100 pg/ml cleared homogenate from the injured (SCI-h) or naive (NaTve-h) spinal cord for 30 days. Cells were then fixed for 20 min with 4% paraformaldehyde in phosphate-buffered saline (PBS) and 40% sucrose at room temperature.
  • PBS phosphate-buffered saline
  • a membrane-based antibody array (Abeam, #ab134002) was used to compare the expression of 41 growth factors in conditioned medium prepared from cells.
  • fbNPC and spNPCs were seeded at an equivalent density of 1 x 10 6 cells on 10 cm laminin coated plates, and after 18 h the conditioned media were collected. The conditioned media was incubated with the antibody array and developed with biotin-conjugate antibodies and HRP-Streptavidin, as per the manufacturer's instructions.
  • fbNPC and spNPCs were plated in their respective maintenance media w/wo NaTve-h or SCI-h (100 pg/ml) at a clonal density of 10 cells/ L in a final volume of 500 l medium in uncoated 24-well plates (Nunc, Rochester, NY). Neurospheres > 50 m in diameter were quantified after ? days of undisturbed culture. Just prior to imaging, the content of each well was transferred to a Matrigel coated dish, incubated for 30 min and fixed with 4% PFA.
  • fbNPC and spNPCs were plated on laminin coated 96-well tissue culture plates (removable strip plates, Corning) at a density of 1 x10 3 cells/100 pl/well and the cell number was determined at 12 , 24, 48 and 72 hours after plating using a Brdll cell proliferation assay (Abeam, #ab 126556), as recommended by the manufacturer.
  • Gel foam (Ferrosan, Denmark) was placed on the spinal cord at the end of the surgical procedure and the incision was closed in layers using standard silk sutures. Animals were allowed to recover in their cage under a heat-lamp and, subsequently, were housed in a 12-hour light-dark cycle at 26°C with free access to food and water. Animals received extensive postoperative care, including Clavamox in drinking water 3 days before injury until the study endpoint. Animals were administered buprenorphine (0.1 mg/kg) for 3 days and meloxicam (1 .0 mg/kg) for 3 days. Injured rats were administered fluids and nutritional support, and their bladders were manually voided three times daily for 14 days as needed.
  • the Accutase was then neutralized by media, cells were detached and spun at 400g for 4 mins, re-suspended in culture media and viable cells counted.
  • Cells were transplanted intraspinally at a density of 50,000 cells/pl.
  • 2 pl of cell suspension were injected per site and there were four injection sites per rat (1 .0 mm bilateral to the midline at 2 mm rostral and caudal). Injections were delivered at 0.6 pl /minute, left to dwell for 2 minutes, and retracted over 2 additional minutes using a Hamilton syringe and a stereotaxic injection system (System UMP3 with Micro4, World Precision Instruments, Sarasota, FL).
  • the control animals also received the same number of injections to the spinal cord with only culture media.
  • LFB Luxol Fast Blue
  • H&E Hematoxylin & Eosin
  • a blinded investigator performed LFB and H&E analyses on tissue ⁇ 2,440 pm centered at the injury epicenter. Unbiased measurements were made using a Cavalieri volume probe from Stereo Investigator (MBF, Bioscience, Wilson, VT) to produce area and volume estimations of preserved white matter and lesional tissue(Wilcox et al., 2014). Lesional tissue was defined as areas with the following aberrant histology; small round cysts, irregularly shaped vacuoles, disorganization of both white and gray matter and eosinophilic neurons. Calculations and analyses were done for tissue sections every 240 pm.
  • VHRUS very high resolution ultrasound
  • VHRUS and Power Doppler imaging were performed as previously described(Soubeyrand et al., 2014). Under isoflurane anesthesia, animals were placed within a custom-made stabilization frame on the imaging platform (Vevo imaging station, Visualsonics, Toronto, Canada). The injury was re-exposed through a mid-line incision and ultrasound gel (Scanning Gel, Medi-lnn, Canada) was placed on the dura mater. The spinal cord was scanned with the VHRUS probe (44 MHz, Vevo 770, Visualsonics, Toronto, Canada). The 3D B-mode scans were analyzed using Imaged software and the TrakEM2 plugin to generate a reproducible cavity volume.
  • Doppler analysis For Power Doppler analysis, a field-of-view with fixed area was defined and centered manually on the central sagittal slice. The Doppler signal was binarized through image thresholding, and batch analysis was carried out for all sagittal sections. The area fraction of positive Doppler signal was multiplied to the actual image area to yield a Doppler-positive area per sagittal slice, which was ultimately summed to yield the total Doppler area for each spinal cord (termed “functional vascularity”). For clarity, all values have been normalized to the sham injury Doppler signal.
  • synaptic density For the analysis of the synaptic density and the ratio of asymmetrical/symmetrical synapses, sets of micrographs for 4 rats in the control-hiPSC-NPC group and 4 rats in the GDNF-hiPSC-NPC group were used.
  • the synaptic counts were expressed as the number of synapses on a membrane length unit of 100 pm.
  • the estimate of synaptic parameters was derived from 145 synapses in hiPSC-NPC and 185 synapses in the GDNF- hiPSC-NPC group.
  • Electrophysiological recordings were carried out under isoflurane anesthesia (1.0- 1.5% inspiratory concentration). Twisted bipolar stimulation electrodes were made with polyimide- insulated stainless-steel wires with outer diameter of 0.2 mm and electrode-tip spacing of 0.1 mm (Plastics One, Roanoke, VA, USA). An aCSF-filled glass microelectrode with a 100 ⁇ 120 pm tip diameter was used for recordings. Stimulation was applied at spinal cord segment T1 at 1.2 mm depth, targeting the dorsal corticospinal tract (dCST).
  • dCST dorsal corticospinal tract
  • Stimulation protocol consisted of delivering a cathodic rectangular wave with a pulse width of 0.1 ms and an amplitude of 1.5 mA, every 10 seconds. Stimulation pulses were generated using a PSIU6 stimulus isolation unit Grass S88 stimulator (Grass Technologies, USA). Evoked compound action potentials (CAPs) were recorded from spinal cord segment C4 at 1.2 mm depth, also targeting the dCST (Li et al., 2016). The spacing between the stimulating and recording electrodes was 10 mm.
  • CAPs were recorded in DC mode with Axoprobe 1A amplifier (Molecular Devices, CA, USA) and processed using pClamp8 software and Digidata 1320A (Molecular Devices, CA, USA) with the sampling rate of 83.33 kHz. 2kHz low-pass and 100 Hz high pass filters were used for these recordings.
  • the data analysis was performed offline using a custom written program in Matlab (Math Works, Natick, MA, USA) to measure the peak-amplitude and peak-latency of the evoked CAP responses in each animal.
  • CAP conduction velocity was calculated by dividing the electrodes’ spacing (10 mm) by the measured peak-latency.
  • Results are stated as mean ⁇ standard error of the mean (SEM) or Standard deviation (SD) as indicated in the figure legends.
  • Neurosphere assay and immunohistological data were analyzed using Student’s t-tests. Histomorphometric and behavioral data were analyzed using two- way analysis of variance (ANOVA) with Tukey’s post hoc test, or one-way ANOVA with Turkey’s post hoc test as indicated in figure legends. The significance level of all analyses was set at p ⁇ 0.05. Data were analyzed with Prism 6 (GraphPad Software, San Diego, CA).
  • Spinal -NPCs will be dissociated into a single-cell suspension at a concentration ranging from 1 x 10 A 4 to 1 x 1O A 6 cells/pl in any vehicle (can be saline or any FDA approved vehicle) or cell can be encapsulated in a biomaterial/ scaffold or matrix to fill in the lesion cavity. Cells will be stereotactically injected into spinal cord in defined rate
  • iNPCs neural progenitor cells
  • CXCL12 N-terminal end is sufficient to induce chemotaxis and proliferation of neural stem/progenitor cells.
  • Delta-Notch signaling controls the generation of neurons/glia from neural stem cells in a stepwise process. Development 130, 1391-1402.
  • Lu P., Wang, Y., Graham, L., McHale, K., Gao, M., Wu, D., Brock, J., Blesch, A., Rosenzweig, E.S., Havton, L.A., et al. (2012). Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell 150, 1264-1273.
  • Immunoglobulin G (IgG) attenuates neuroinflammation and improves neurobehavioral recovery after cervical spinal cord injury. J. Neuroinflammation 9.
  • Notchl and Notch3 instructively restrict bFGF-responsive multipotent neural progenitor cells to an astroglial fate. Neuron 29, 45-55.
  • Neural precursor cell transplantation enhances functional recovery and reduces astrogliosis in bilateral compressive/contusive cervical spinal cord injury.
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